Acrosin (E.N. 3.4.21.10) is a unique trypsin-like enzyme present in mammalian sperm acrosomes and is used by sperm to digest a path through the zona pellucida of the ovum, a prerequisite to fertilization. This project is designed to continue the active site characterization of acrosin by defining the topography (structure) of acrosin's active site so that specific affinity labeling (irreversible) acrosin inhibitors can be synthesized which may be used to inhibit fertilization. Particular emphasis will be placed on determining the mechanism of acrosen's unique catalytic properties (i.e., stimulation and stabilization by polyamines, inhibition with various arginine analogs, etc.) in order to define reagents that specifically bind to acrosin and not trypsin (the enzyme most similar to acrosin). Agents that specifically bind to acrosin will be modified to become affinity labeling inhibitors. These inhibitors will be tested as to their specificity for acrosin and the functional amino acid residues which participate in the binding will be determined. The possible involvement of numerous physiological factors and the biological activity (inhibition of sperm bound acrosin) will be asscertained. Another aim of this project is to use radioactive affinity labeling inhibitors to define the physiologically significant form(s) of acrosin and to characterize the unique properties of this (these) form(s). Also, since the physiologically active acrosin is membrane bound, artificial membranes in the form of phospholipid micelles (liposomes) will be utilized as a model system to study various parameters of membrane bound acrosin which may affect the design of the inhibitors. Finally the most significant experiments will be repeated with human acrosin and sperm to permit an accurate evaluation of the possible contraceptive usefulness of these inhibitors.